U.S. patent application number 10/170209 was filed with the patent office on 2003-01-16 for electro-optical detection device.
Invention is credited to Saraf, Ravi.
Application Number | 20030013185 10/170209 |
Document ID | / |
Family ID | 26971211 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030013185 |
Kind Code |
A1 |
Saraf, Ravi |
January 16, 2003 |
Electro-optical detection device
Abstract
A device for detecting molecular binding events rapidly and at
very high sensitivity is provided. The device molecular binding
material (such as DNA or protein) positioned in a conductive path
between electrodes. Binding of a ligand to the binding material is
detected as a change in a frequency response in applied oscillatory
field. The device may also contain an optical measurement system
(based, for example, on a laser) which detects the change in
thickness of the molecular binding material under an applied
oscillatory field with and without a bound material of
interest.
Inventors: |
Saraf, Ravi; (Blacksburg,
VA) |
Correspondence
Address: |
WHITHAM, CURTIS & CHRISTOFFERSON, P.C.
11491 SUNSET HILLS ROAD
SUITE 340
RESTON
VA
20190
US
|
Family ID: |
26971211 |
Appl. No.: |
10/170209 |
Filed: |
June 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60299416 |
Jun 21, 2001 |
|
|
|
60368956 |
Apr 2, 2002 |
|
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Current U.S.
Class: |
435/287.2 ;
205/777.5 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 27/3276 20130101 |
Class at
Publication: |
435/287.2 ;
205/777.5 |
International
Class: |
C12M 001/34; C12Q
001/00 |
Goverment Interests
[0002] This invention was made using funds from grants from the
Naval research foundation having grant number N00014-01-1-0977. The
government may have certain rights in this invention.
Claims
We claim:
1. A device for detecting molecular binding events, comprising: a
capacitor with at least two spaced apart electrodes; a molecular
binding material positioned in a conductive path between said at
least two spaced apart electrodes; means for applying an
oscillatory field on said molecular binding material using said
capacitor; and means for detecting binding of a material of
interest to said molecular binding material based on changes in a
frequency response of said molecular binding material under an
applied oscillatory field.
2. The device of claim 1 wherein said means for detecting binding
comprises an optical measurement system which detects a first
change in thickness of said molecular binding material under an
applied oscillatory field without a bound material of interest and
a second change in thickness of said molecular binding material
under an applied oscillatory field with a bound material of
interest, wherein differences between said first and second change
in thickness indicate binding of said material of interest.
3. The device of claim 2 wherein said optical measurement system
comprises a laser focused on said molecular binding material, a
detector for detecting laser light which has passed through or been
reflected by said molecular binding material and a control laser
light which has not passed through or been reflected by said
molecular binding material, and instrumentation for determining a
thickness of said molecular binding material based on differences
in measurements between said laser light and said control laser
light.
4. A device for detecting molecular binding events, comprising: a
substrate; a plurality of capacitors formed on said substrate, each
of said capacitors having at least two spaced apart electrodes; a
molecular binding material positioned in a conductive path between
each of said at least two spaced apart electrodes; means for
applying an oscillatory field on said molecular binding material
using said capacitor; and means for detecting binding of a material
of interest to said molecular binding material based on changes in
a frequency response of said molecular binding material under an
applied oscillatory field.
5. The device of claim 4 wherein said means for detecting binding
comprises an optical measurement system which detects a first
change in thickness of said molecular binding material under an
applied oscillatory field without a bound material of interest and
a second change in thickness of said molecular binding material
under an applied oscillatory field with a bound material of
interest, wherein differences between said first and second change
in thickness indicate binding of said material of interest.
6. The device of claim 5 wherein said optical measurement system
comprises a laser focused on said molecular binding material, a
detector for detecting laser light which has passed through or been
reflected by said molecular binding material and a control laser
light which has not passed through or been reflected by said
molecular binding material, and instrumentation for determining a
thickness of said molecular binding material based on differences
in measurements between said laser light and said control laser
light.
7. The device of claim 4 wherein said molecular binding material
includes a plurality of different materials, each of said different
materials being positioned between spaced apart electrodes of
different capacitors of said plurality of capacitors.
8. A method for detecting molecular binding events, comprising the
steps of: depositing a sample between spaced apart electrodes of a
capacitor, wherein said capacitor includes a molecular binding
material positioned in a conductive path between said at least two
spaced apart electrodes; applying an oscillatory field on said
molecular binding material using said capacitor; and detecting
binding of a material of interest in said sample to said molecular
binding material based on changes in a frequency response of said
molecular binding material under an applied oscillatory field.
9. The method of claim 8 wherein said detecting step includes
detecting a first change in thickness of said molecular binding
material under an applied oscillatory field without a bound
material of interest and a second change in thickness of said
molecular binding material under an applied oscillatory field with
a bound material of interest, wherein differences between said
first and second change in thickness indicate binding of said
material of interest.
Description
[0001] This application claims benefit of U.S. provisional patent
application serial No. 60/299,416 filed Jun. 21, 2002, and U.S.
provisional patent application serial No. 60/368,956, filed Apr. 2,
2002, the complete contents of which are hereby incorporated by
reference.
DESCRIPTION
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention generally relates to an electro-optical device
for the detection of molecular binding events. In particular, the
invention provides an electro-optical device that measures the
change in an electromechanical property of an immobilized molecule
as it is exposed to a bioagent or other chemical, and methods for
its use.
[0005] 2. Background of the Invention
[0006] The detection of molecular binding events forms the basis of
many fundamental investigations with wide-ranging applications,
such as genetic studies and drug discovery. For example, the
decipherment of DNA sequences is very important for the diagnosis
of diseases, for drug design and in fostering the understanding of
various biological mechanisms. Traditional methods involve
"reading" a gene sequence base-pair by base-pair. DNA "chip"
technologies have provided methods whereby several base-pairs can
be read simultaneously. Further, this technology is a combinatorial
approach in which 10's or even 100's of gene sequences can be read
at the same time. However, these methods rely on tagging the DNA
with a fluorescent dye to facilitate detection. This requires the
utilization of labeling protocols, and results in chemical
modification of the DNA, i.e. the DNA bears a fluorescent dye
molecule.
[0007] Similarly, the analysis of proteins, for example in drug
design, may require the investigation of enzymatic activity by
probing the binding of substrates and inhibitors of an enzyme. In
probing certain large proteins (such as immunoglobulins) some
spectroscopic methods are available where no tagging is required
(e.g. Surface Plasmon Resonance Spectroscopy). However, these
techniques are not useful for the analysis of smaller proteins or
for understanding the roles of specific amino acid sequences due to
a lack of sensitivity, and other means, such as labeling, must be
resorted to in order to detect binding products. Again, this
necessitates the use of labeling protocols and results in the
chemical modification of whichever component of the system is
labeled.
[0008] It would be highly beneficial to have available methodology
that would allow the quantitative analysis of small biomolecular
binding events such as ssDNA hydridization and protein-protein and
protein-nucleic acid interactions, without the necessity of
chemically modifying the reactants by labeling.
SUMMARY OF THE INVENTION
[0009] The present invention provides a device for detecting
molecular binding events rapidly and at very high sensitivity. The
device comprises a capacitor with at least two spaced apart
electrodes; a molecular binding material positioned in a conductive
path between the electrodes; a means for applying an oscillatory
field to the molecular binding material using the capacitor; and a
means for detecting binding of a material of interest to the
molecular binding material. The detection is based on changes in a
frequency response of the molecular binding material under the
applied oscillatory field.
[0010] The device may also contain an optical measurement system
which detects the change in thickness of the molecular binding
material under an applied oscillatory field with and without a
bound material of interest. Differences between the change in
thickness with and without a bound material indicate that binding
of the material of interest has occurred.
[0011] The optical measurement system may include a laser focused
on the molecular binding material, a detector for detecting
reflections from first and second opposing sides of said molecular
binding material, and instrumentation for determining the thickness
of the molecular binding material based on reflections detected by
the detector.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. A schematic representation of the electro-optical
device of the present invention.
[0013] FIG. 2A and B. A, monolayer of biomolecules on an inert,
solid substrate. The monolayer may be a covalently bonded,
self-assembled or simply adsorbed thin film. The film thickness at
electric field E=0 is d. B, as the electric field is applied
parallel to the substrate surface, the molecule in the film orient
causing a change in film thickness by .DELTA.d, due to the Poisson
effect.
[0014] FIG. 3. Set-up of the differential interferometer.
[0015] FIG. 4. Schematic of the signal from the spectrum analyzer.
The x-axis is frequency and the y-axis is amplitude.
[0016] FIG. 5. Schematic of a biochip to perform combinatorial
analysis.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0017] The present invention provides an electro-optical device for
detecting and measuring changes in a macromolecule, and methods for
its use. In a preferred embodiment, the change that is detected is
the result of ligand binding. The device does not require the use
of chemical labels; thus, the molecules that are detected can
remain in an unmodified state, i.e. the method is
non-destructive.
[0018] The construction of the device takes advantage of the
piezoelectric properties of macromolecules when they are placed in
an electric field. A schematic representation of a preferred
embodiment of the device of the present invention is given in FIG.
1. With reference to FIG. 1, the device comprises a substrate 13
having a thermal layer 11 disposed thereon. Electrodes 12 are
located on a top surface 15 of said thermal layer 11. The
electrodes are separated by a space 16 that is wide enough to allow
placement of a sample layer or film 10 therebetween. The sample
film 10 may be deposited between the electrodes 12, fully covering
the space 16 between the electrodes 12. The layer or film may be
located between the electrodes and extend past the space 16 to
conformally coat the electrodes as well. The device further
comprises a power source 14 which modulates an electric field at
frequency .omega..sub.s. Only the film between the electrodes
experiences the electric field. While FIG. 1 shows a pair of
capacitor plates as electrodes 12, it should be understood that a
comb configured capacitor arrangement may be used in the practice
of this invention.
[0019] FIG. 2 illustrates the central principle of the device of
the present invention. The polymeric macromolecules deposited on
the device of the present invention are "mechanically soft" (i.e.
non-rigid, deformable) dielectric polymers with high permanent
dipole moments or polarizability. When an electric field is applied
to a thin film of such polymers, they orient along the direction of
the field, causing a change in the thickness of the film. FIG. 2A
depicts a polymer film 20 film deposited on the thermal layer 11
disposed on a substrate 13. In the absence of an applied electric
field (E=0, FIG. 2A), the polymer film exhibits a thickness d. When
an electric field is applied to the polymer film 20 (E>0, FIG.
2B) the polymers orient themselves along the field direction,
thereby changing the thickness of the film by .DELTA.d. If the
field is oscillatory, at frequency .omega..sub.s, the thickness
will modulate at .omega..sub.s (linier effect due to Piezoelectric
behavior) and at 2.omega..sub.s (non-linear effect due to
electrostrictive behavior. For the linier effect the change in
thickness, .DELTA.d, is proportional to the electric field E. For
the non-linier effect the change in .DELTA.d is proportional to
E.sup.2. Thus, if the applied electric field is Ecos(.omega.t) then
.DELTA.d will be proportional to Ecos(.omega.t), or in other words
the thickness will modulate at frequency .omega. (piezoelectric
effect). On the other hand, for the electrostrictive effect,
.DELTA.d will be proportional to cos.sup.2(.omega.t) causing the
thickness to modulate at cos(2.omega.t) due to the relationship
that states, cos.sup.2(.omega.t)={fraction (1/2)}(1+cos(2.omega.t).
The frequency response of the film i.e. .DELTA.d as a function of
.omega..sub.s, will depend on the coupling between the mechanical
property and electronic property of the film. The electromechanical
coupling will depend on the conformation, environment, and
structure of the individual molecules of the film. Further, when
the polymers which make up the film undergo a change (e.g. when a
ligand is bound) the frequency response of the film (.DELTA.d as a
function of .omega..sub.s) will change. The device and methods of
the present invention are designed to detect and measure this
change.
[0020] FIG. 3 illustrates a setup of a differential interferometer
incorporating the device of the present invention. With reference
to FIG. 3, a device of the present invention 30 with a sample film
31 deposited thereon is shown. The sample is placed under a laser
beam from a laser 33. A portion of the beam is diverted via a beam
splitter 34 to an acusto-optical device 35 to create a
.omega..sub.b phase modulated reference beam. The other portion
travels into the sample film 31 and is reflected back into the
sample film again by the substrate 30. As the sample thickness
changes due to modulation caused by the electric field, the travel
distance of the laser beam changes by an amount .DELTA.(nd), where
n is the refractive index of the beam. This modulation causes a
phase modulation of this portion of the laser beam by
(4.pi./.lambda.)(.DELTA.(nd)), where .lambda. is the wavelength of
the laser light. Since (.DELTA.(nd) modulates at frequencies
.omega. and 2.omega., the phase modulates at the same frequency.
Upon mixing the sample beam with the reference beam into a detector
36, a frequency-modulated signal is produced. The frequency
distribution is analyzed in a spectrum analyzer 37 that will show a
strong peak at .omega..sub.b and satellite peaks at
.omega..sub.b+/-n.omega. Usually only n=1 and n=2 are observed.
[0021] A schematic spectrum as seen on the spectrum analyzer is
depicted in FIG. 4. The main peak in the center is at .omega..sub.b
and has am amplitude of A.sub.0. The satellite peaks at
.omega..sub.b+/-.omega. are of nominally the same amplitude
A.sub.1, and the satellite peaks at .omega..sub.b+2.omega. are of
nominally the same amplitude A.sub.2. The ratio of powers with
respect to the main peak to a very good approximation (within less
than 0.5%) is given as:
R.sub.1=(A.sub.1/A.sub.0)(2.pi./.lambda.)(.DELTA.(nd).sub.I
[0022] and
R.sub.2=(A.sub.2/A.sub.0)(.pi./.lambda.)(2.DELTA.(nd).sub.II
[0023] where (.DELTA.(nd).sub.I and .DELTA.(nd).sub.II are
thickness modulation at frequencies .omega. and 2.omega.. From the
above equations, since R.sub.1 and R.sub.2 are measured, the
thickness modulation can be determined. The material property
(i.e., .DELTA.(nd).sub.I and .DELTA.(nd).sub.II) in the device can
be measure as a function of experimental conditions such as:
.omega..sub.b, .omega., electric field amplitude, and temperature.
Both the magnitude and dependence of the material properties on the
experimental conditions will be sensitive to the molecules
constituting the film. Thus, by measuring .DELTA.(nd).sub.I and
.DELTA.(nd).sub.II before and after a bioreaction (e.g. a
conformational change, or ligand binding) the occurrence of that
particular event can be probed.
[0024] Due to the change in mass and conformation, the frequency
spectrum of the film will change.
[0025] In a preferred embodiment of the present invention, the
substrate portion of the device is made from a highly reflective
material. In a preferred embodiment, the substrate is comprised of
silicon (Si). However, those of skill in the art will recognize
that other materials may also be utilized to form the substrate,
including but not limited to polymers and biomolecules. In a
preferred embodiment, the thermal layer is comprised of SiO.sub.2.
However, those of skill in the art will recognize that other
materials may also be utilized to form the substrate layer,
including but not limited to Au or Ag coated solid substrate such
as glass, ceramic or polymer.
[0026] Methods of constructing such substrates and thermal layers
are well known to those in the art.
[0027] In the device of the present invention, electrodes are
located on a top surface of the substrate. In a preferred
embodiment of the present invention, the electrodes are gold
electrodes. However, those of skill in the art will recognize that
other appropriate materials exist which may also be utilized to
form the electrodes, including but not limited to Ag, Pt, metal
alloy of noble metal, Indium-Tin-Oxide, etc.
[0028] Those of skill in the art are well-acquainted with
techniques for forming electrodes on a substrate. Such methods
include but are not limited to sputter deposition, vapor
deposition, electro plating, electroless plating, inkjet printing,
etc.
[0029] In the device of the present invention, a meso-scale layer
or film of immobilized polymeric molecules is located on a top
surface of the device. The polymers which are deposited on the
device of the present invention are, in general, macromolecular in
nature, i.e. they are in the size range of from about 100 microns
to about 1 nm. The polymers have intrinsically high permanent
dipole moments, or are polarizable, and they exhibit piezoelectric
and electrorestrictive properties. The molecules may be capable of
binding a ligand. Polymers which may be deposited as a layer or
film in the practice of the present invention include but are not
limited to nucleic acids (e.g. ssDNA, dsDNA, ssRNA, dsRNA, PNA),
proteins (e.g. enzymes, antibodies), lipids, polysaccharides, etc.
In a preferred embodiment, the polymer is DNA. In yet another
preferred embodiment, the polymer is a protein.
[0030] By "deposited as a layer or film" we mean that a layer of
molecules is attached to an upper surface of the device, e.g. to
the thermal layer, and optionally, to the electrodes. The
attachment may be effected by any of many suitable means which are
well-known to those of skill in the art, including but not limited
to covalent, ionic, hydrophobic bonding, adsorption, self assembly
(Reference: Y Xia, J. A. Rohers, K. E. Paul, G. M. Whitesides,
Unconventional Methods for Fabricating and Patterning
Nanostructures, Chem. rev. Vol. 99, pages 1823-1848 (1999). The
means of carrying out such attachments are well-known to those of
skill in the art. For example, an SiO.sub.2 thermal layer can be
functionalized to contain hydroxyl groups using a standard Piranha
solution, followed by silane treatment to obtain amine, carboxylic
or other reactive groups. These reactive groups can then be
utilized to bind functional groups on the molecules to be attached,
e.g. with hydroxyl or phosphate groups of nucleic acids, with amine
or carboxylic acid groups of proteins, by hydroxyl groups of
saccharides, and the like. The density of the polymers in the film
will be on the order of about 1 gm/ml. Further, the molecule may be
attached directly to the top surface of the device, or may be
attached via a polymeric linker or spacer. In a preferred
embodiment, a single type of molecule is deposited on the device.
However, for certain applications it may be desired to deposit two
or more different molecules, for example, in a microarray where
different types of molecules will be immobilized at different areas
of the same substrate.
[0031] By "capable of binding a ligand" we mean that the polymers
that comprise the film are capable of binding to another molecule
of interest, i.e. they may bind to a ligand. Therefore, one
potential change that may be detected by the device and methods of
the present invention is the binding of a ligand by the polymers
that comprise the film. Ligands which may bind to the polymers
include but are not limited to complementary ssDNA, dsDNA,
complementary ssRNA, dsRNA, proteins, polypeptides, lipids,
saccharides, various protein substrates and inhibitors, co-factors,
metals, toxic substances, small organic molecules (e.g. molecular
weight less than about 100), drugs, disease producing entities
(e.g. viruses, bacteria and other pathogens, or components
thereof), antibodies, etc.
[0032] The source of such ligands may be any of a wide variety of
sources, including but not limited to biological samples such as
blood, urine, etc.; environmental samples such as water from
reservoirs or waste water; comestible items; and the like. Further,
the device of the present invention may function in either a liquid
environment, or in air.
[0033] In another embodiment of the invention, changes other than
the binding of a ligand are detected by the device and methods of
the present invention. For example, changes in the conformation of
an immobilized macromolecule as a result of an alteration in the
environment of the macromolecule may also be detected. Such
alterations include but are not limited to changes in pH,
temperature (e.g. to detect denaturation and/or renaturation,
sensitivity to cold, etc), ionic strength, etc.
[0034] Further, the detection of the impact of an alteration in the
environment on the binding of a ligand may be detected. For
example, the ability of a macromolecular polymer to bind a ligand
at different pH values, at different ionic strengths, at different
temperatures, or in the presence of other effector molecules, may
also be detected by the device and methods of the present
invention. Those of skill in the art will recognize that any
alteration in the polymeric macromolecules deposited on the device
of the present invention may be detected by the device and methods
of the present invention, so long as the alteration results in a
measurable change in the piezoelectric properties of the polymers
in the film.
[0035] In one embodiment of the invention, the change may be
induced by ligand binding. By "ligand binding" we mean that a
ligand has become associated with polymers in the polymer film. The
association may be irreversible or reversible, and may be the
result of binding via, for example, covalent, ionic, or hydrophobic
forces. If the association is reversible, the affinity of the
ligand for the polymer will generally be on the order of about 1%
to about 100%.
[0036] Binding events which may be detected by the device and
methods of the present invention include but are not limited to
nucleic acid hybridization (e.g. complementary ssDNA and/or ssRNA
binding); protein-ligand binding (e.g. protein-substrate or
protein-inhibitor binding); the binding of regulatory factors to a
macromolecule such as a protein; and the like.
[0037] With respect to ligand binding, the values of the amount of
change that is detected will depend on the "perfection" of binding.
For example, for ssDNA hybridization, the location of one or more
base-pair mismatches can be detected. Thus, by measuring the above
ratios, subtle variations in sequence (for example, those caused by
mutations) can be determined. Although in principle the ratios may
be estimated theoretically for various sequences with corresponding
mismatches, the values may more simply be obtained by experimental
calibration. In this case, ratios of R1 and R2 may be determined
experimentally, and a database for known sequences with a
predetermined number and location of mismatches may be established
from the data.
[0038] The device and methods of the present invention detect
molecular binding events rapidly (the data acquisition response
time is<1 minute) and at extremely low sensitivity.
[0039] In one embodiment of the present invention, the device of
the present invention is integrated into a chip format that allows
combinatorial analysis. Such an arrangement is depicted in FIG. 5
where parallel electrode lines 40 of width w can be deposited at
spacing s and patches of sample film 41 (pixels) can be deposited
between the electrode lines 40. An electric field is applied and a
laser beam is scanned over various locations on the chip to probe
the frequency response to the electric field. The patches of sample
film 41 may be the same (for example, to provide control sections
on the chip) or different (for example, so that many different
molecules, such as ssDNA sequences, may be assayed on a single
chip). The minimum value of s will be determined by the size of the
laser beam, and in general, pixel size will be in the range of
about 10 by 10 .mu.m or less. Thus, a 1 centimeter square chip with
interconnection pads,(i.e. pads that connect the electrode to the
power supply, which can themselves be electrodes) can readily
contain more than 3,000 pixels, i.e. can contain more than 3000
different patches of immobilized molecules. For a typical DNA chip,
the pixel size is determined by the size of the probe beam.
Typically, for a probe beam from an He--Ne laser with .lambda.=633
nm, the pixel may be about 5 by 5 .mu.m.
[0040] Those of skill in the art will recognize that the device and
methods of the present invention may be utilized for the detection
of numerous substances in a wide variety of fields. For example,
the device and methods are useful in fields of drug discovery to
identify compounds that bind to macromolecules (e.g. inhibitors of
an enzyme); or to accomplish DNA sequencing via hybridization of
ssDNA of varying sequences; or to detect pollutants, toxins and
other noxious substances, for example, in biological warfare.
EXAMPLES
Example 1
Detection of ssDNA Hybridization
[0041] Two electrodes are established a short distance (e.g. 10
microns) apart. Between the electrodes, a film of ssDNA of
relatively short length (e.g. 25 bp) is immobilized and kept in an
extended form via "optical tweezers". An electric field is applied
between the electrodes, causing the DNA to become a charged
material in which one side will be positive and will move toward
the negative electrode. Upon reversing the polarity of the field,
the DNA will appear to "dance" in a similar fashion to long grass
waving in the wind, resulting in a measurable change in the
thickness of the film. The electric field is oscillated (vibrated)
at different frequencies. At certain frequencies, the motion of the
DNA is greater because it is at resonance at those frequencies.
[0042] The ssDNA resembles a piece of rope, with its vibrational
behavior dependent on the rigidity of the rope. If a binding event
occurs, such as the hybridization of a complementary strand of
ssDNA, the rope will be substantially thicker and more rigid than
the ssDNA. This causes a change in the resonance frequency of the
molecules in the film. When such a change is detected, it is
indicative of a binding event.
[0043] Measurements as precise as to the order of a single base
pair are made and the resulting frequency changes are used to
quantify the degree of hybridization of the known ssDNA sequence.
For example, if a known ssDNA of 25 bp joins to complementary ssDNA
in a biological sample, if of the 25 bp 24 are exactly matched but
1 is mismatched, the structure is slightly different than if all 25
bp are exactly matched. Therefore, the oscillation of the 24 out of
25 bp matched double helix (or any other combination, e.g. a 23 out
of 25 bp match) differs from that of the perfect 25 bp match (or
from any other bp match combination.) Thus, the degree of
hybridization is detected by detecting the corresponding changes in
frequency. Moreover, because changes in vibration on the order of
0.5 angstroms are detected, and a bp is about 3 angstroms in size,
the device of the present invention also locates unbound sites such
as at the end or middle of the known ssDNA sequence.
[0044] While the invention has been described in terms of its
preferred embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims. Accordingly, the present
invention should not be limited to the embodiments as described
above, but should further include all modifications and equivalents
thereof within the spirit and scope of the description provided
herein.
* * * * *